Technical Abstract:
We have been exploring the use of genetically engineered anthocyanin over-production as a visual indicator of gene activation and silencing in plants. Previous work demonstrated that constitutive over-expression of genes encoding specific Arabidopsis transcription factors from the myb family can produce ectopic over-production of anthocyanin pigments in Arabidopsis and several other plant species (Xia, et. al [2000], The Plant Cell,12:2383). We have independently generated several transgenic N. tabacum lines that constitutively over-express the Arabidopsis PAP2, or AtMyb90, gene (CaMV35S::AtMYB90) and several of the resulting transgenic lines display extensive anthocyanin pigmentation in nearly all tissues, but are otherwise identical to wild type plants (except for a somewhat reduced growth rate). Transgene silencing, as indicated by a clear reduction in pigment production and a concomitant drop in steady state transgene mRNA levels, can be induced by Agrobacterium infiltration that generates hairpin RNA covering most of the AtMyb90 coding region. Silencing is observed both locally, at the infiltration site, and systemically in newly developing leaves above the infiltrated leaves. One line, Myb27, was found to show similar patterns of pigment loss (and mRNA reduction) when transgene dosage was doubled (homozygous at the transgene locus). Hemizygous Myb27 plants are dark purple, as opposed to homozygotes that show only limited anthocyanin pigmentation. The loss of pigmentation within homozygous Myb27 plants correlates with reduced transgene mRNA levels and appears to be impacted by developmental and/or environmental (light) factors. Work addressing the nature of smRNAs with homology to the AtMyb90 transgene is presented separately.